Armored TGFßRIIDN ROR1-CAR T cells reject solid tumors and resist suppression by constitutively-expressed and treatment-induced TGFß1.

BACKGROUND: Chimeric antigen receptor (CAR) T-cell therapy target receptor tyrosine kinase-like orphan receptor 1 (ROR1) is broadly expressed in hematologic and solid tumors, however clinically-characterized ROR1-CAR T cells with single chain variable fragment (scFv)-R12 targeting domain failed to induce durable remissions, in part due to the immunosuppressive tumor microenvironment (TME). Herein, we describe the development of an improved ROR1-CAR with a novel, fully human scFv9 targeting domain, and augmented with TGFßRIIDN armor protective against a major TME factor, transforming growth factor beta (TGFß). METHODS: CAR T cells were generated by lentiviral transduction of enriched CD4+ and CD8+ T cells, and the novel scFv9-based ROR1-CAR-1 was compared with the clinically-characterized ROR1-R12-scFv-based CAR-2 in vitro and in vivo. RESULTS: CAR-1 T cells exhibited greater CAR surface density than CAR-2 when normalized for %CAR+, and produced more interferon (IFN)-γ tumor necrosis factor (TNF)-α and interleukin (IL)-2 in response to hematologic (Jeko-1, RPMI-8226) and solid (OVCAR-3, Capan-2, NCI-H226) tumor cell lines in vitro. In vivo, CAR-1 and CAR-2 both cleared hematologic Jeko-1 lymphoma xenografts, however only CAR-1 fully rejected ovarian solid OVCAR-3 tumors, concordantly with greater expansion of CD8+ and CD4+CAR T cells, and enrichment for central and effector memory phenotype. When equipped with TGFß-protective armor TGFßRIIDN, CAR-1 T cells resisted TGFß-mediated pSmad2/3 phosphorylation, as compared with CAR-1 alone. When co-cultured with ROR-1+ AsPC-1 pancreatic cancer line in the presence of TGFß1, armored CAR-1 demonstrated improved recovery of killing function, IFN-γ, TNF-α and IL-2 secretion. In mouse AsPC-1 pancreatic tumor xenografts overexpressing TGFß1, armored CAR-1, in contrast to CAR-1 alone, achieved complete tumor remissions, and yielded accelerated expansion of CAR+ T cells, diminished circulating active TGFß1, and no apparent toxicity or weight loss. Unexpectedly, in AsPC-1 xenografts without TGFß overexpression, TGFß1 production was specifically induced by ROR-1-CAR T cells interaction with ROR-1 positive tumor cells, and the TGFßRIIDN armor conferred accelerated tumor clearance. CONCLUSIONS: The novel fully human TGFßRIIDN-armored ROR1-CAR-1 T cells are highly potent against ROR1-positive tumors, and withstand the inhibitory effects of TGFß in solid TME. Moreover, TGFß1 induction represents a novel, CAR-induced checkpoint in the solid TME, which can be circumvented by co-expressing the TGßRIIDN armor on T cells.

Inhibition of Pseudomonas aeruginosa and Mycobacterium tuberculosis disulfide bond forming enzymes.

In bacteria, disulfide bonds confer stability on many proteins exported to the cell envelope or beyond, including bacterial virulence factors. Thus, proteins involved in disulfide bond formation represent good targets for the development of inhibitors that can act as antibiotics or anti-virulence agents, resulting in the simultaneous inactivation of several types of virulence factors. Here, we present evidence that the disulfide bond forming enzymes, DsbB and VKOR, are required for Pseudomonas aeruginosa pathogenicity and Mycobacterium tuberculosis survival respectively. We also report the results of a HTS of 216,767 compounds tested against P. aeruginosa DsbB1 and M. tuberculosis VKOR using Escherichia coli cells. Since both P. aeruginosa DsbB1 and M. tuberculosis VKOR complement an E. coli dsbB knockout, we screened simultaneously for inhibitors of each complemented E. coli strain expressing a disulfide-bond sensitive ß-galactosidase reported previously. The properties of several inhibitors obtained from these screens suggest they are a starting point for chemical modifications with potential for future antibacterial development.

Hydrophobic Residue in Escherichia coli Thioredoxin Critical for the Processivity of T7 DNA Polymerase.

Bacteriophage T7 uses the thioredoxin of its host, Escherichia coli, to enhance the processivity of its DNA polymerase, a requirement for the growth of phage T7. The evolutionarily conserved structure and high degree of homology of amino acid sequence of the thioredoxin family imply that homologues from other organisms might also interact with T7 DNA polymerase to support the phage growth. Despite the structural resemblance, human thioredoxin, whose X-ray crystallographic structure overlaps with that of the E. coli protein, cannot support T7 phage growth. It does not form a complex with T7 DNA polymerase as determined by surface plasmon resonance and thus does not increase the processivity. Homologous scanning analysis using this nonfunctional homologue reveals that the 60 N-terminal and the 12 C-terminal amino acid residues of E. coli thioredoxin can be substituted for its human counterpart without significantly affecting phage growth. Comparison of chimeric thioredoxins, followed by site-directed mutagenesis, identifies leucine 95 as a critical element. This residue may contribute to hydrophobic interactions with the thioredoxin-binding loop of the polymerase; levels of DNA binding and thus nucleotide polymerization are significantly decreased in the absence of this residue. The results suggest that the specific interactions at the interface of thioredoxin and DNA polymerase, rather than the overall structure, are important in the interactions that promote high processivity.

Inhibition of virulence-promoting disulfide bond formation enzyme DsbB is blocked by mutating residues in two distinct regions.

Disulfide bonds contribute to protein stability, activity, and folding in a variety of proteins, including many involved in bacterial virulence such as toxins, adhesins, flagella, and pili, among others. Therefore, inhibitors of disulfide bond formation enzymes could have profound effects on pathogen virulence. In the Escherichia coli disulfide bond formation pathway, the periplasmic protein DsbA introduces disulfide bonds into substrates, and then the cytoplasmic membrane protein DsbB reoxidizes DsbA's cysteines regenerating its activity. Thus, DsbB generates a protein disulfide bond de novo by transferring electrons to the quinone pool. We previously identified an effective pyridazinone-related inhibitor of DsbB enzymes from several Gram-negative bacteria. To map the protein residues that are important for the interaction with this inhibitor, we randomly mutagenized by error-prone PCR the E. coli dsbB gene and selected dsbB mutants that confer resistance to this drug using two approaches. We characterized in vivo and in vitro some of these mutants that map to two areas in the structure of DsbB, one located between the two first transmembrane segments where the quinone ring binds and the other located in the second periplasmic loop of DsbB, which interacts with DsbA. In addition, we show that a mutant version of a protein involved in lipopolysaccharide assembly, lptD4213, is synthetically lethal with the deletion of dsbB as well as with DsbB inhibitors. This finding suggests that drugs decreasing LptD assembly may be synthetically lethal with inhibitors of the Dsb pathway, potentiating the antibiotic effects.

Genetic requirements for sensitivity of bacteriophage t7 to dideoxythymidine.

We previously reported that the presence of dideoxythymidine (ddT) in the growth medium selectively inhibits the ability of bacteriophage T7 to infect Escherichia coli by inhibiting phage DNA synthese (N. Q. Tran, L. F. Rezende, U. Qimron, C. C. Richardson, and S. Tabor, Proc. Natl. Acad. Sci. U. S. A. 105:9373-9378, 2008, doi:10.1073/pnas.0804164105). In the presence of T7 gene 1.7 protein, ddT is taken up into the E. coli cell and converted to ddTTP. ddTTP is incorporated into DNA as ddTMP by the T7 DNA polymerase, resulting in chain termination. We have identified the pathway by which exogenous ddT is converted to ddTTP. The pathway consists of ddT transport by host nucleoside permeases and phosphorylation to ddTMP by the host thymidine kinase. T7 gene 1.7 protein phosphorylates ddTMP and ddTDP, resulting in ddTTP. A 74-residue peptide of the gene 1.7 protein confers ddT sensitivity to the same extent as the 196-residue wild-type gene 1.7 protein. We also show that cleavage of thymidine to thymine and deoxyribose-1-phosphate by the host thymidine phosphorylase greatly increases the sensitivity of phage T7 to ddT. Finally, a mutation in T7 DNA polymerase that leads to discrimination against the incorporation of ddTMP eliminates ddT sensitivity.

Thioredoxin, the processivity factor, sequesters an exposed cysteine in the thumb domain of bacteriophage T7 DNA polymerase.

Gene 5 protein (gp5) of bacteriophage T7 is a non-processive DNA polymerase. It achieves processivity by binding to Escherichia coli thioredoxin (trx). gp5/trx complex binds tightly to a primer-DNA template enabling the polymerization of hundreds of nucleotides per binding event. gp5 contains 10 cysteines. Under non-reducing condition, exposed cysteines form intermolecular disulfide linkages resulting in the loss of polymerase activity. No disulfide linkage is detected when Cys-275 and Cys-313 are replaced with serines. Cys-275 and Cys-313 are located on loop A and loop B of the thioredoxin binding domain, respectively. Replacement of either cysteine with serine (gp5-C275S, gp5-C313S) drastically decreases polymerase activity of gp5 on dA(350)/dT(25). On this primer-template gp5/trx in which Cys-313 or Cys-275 is replaced with serine have 50 and 90%, respectively, of the polymerase activity observed with wild-type gp5/trx. With single-stranded M13 DNA as a template gp5-C275S/trx retains 60% of the polymerase activity of wild-type gp5/trx. In contrast, gp5-C313S/trx has only one-tenth of the polymerase activity of wild-type gp5/trx on M13 DNA. Both wild-type gp5/trx and gp5-C275S/trx catalyze the synthesis of the entire complementary strand of M13 DNA, whereas gp5-C313S/trx has difficulty in synthesizing DNA through sites of secondary structure. gp5-C313S fails to form a functional complex with trx as measured by the apparent binding affinity as well as by the lack of a physical interaction with thioredoxin during hydroxyapatite-phosphate chromatography. Small angle x-ray scattering reveals an elongated conformation of gp5-C313S in comparison to a compact and spherical conformation of wild-type gp5.

Characterization of a nucleotide kinase encoded by bacteriophage T7.

Gene 1.7 protein is the only known nucleotide kinase encoded by bacteriophage T7. The enzyme phosphorylates dTMP and dGMP to dTDP and dGDP, respectively, in the presence of a phosphate donor. The phosphate donors are dTTP, dGTP, and ribo-GTP as well as the thymidine and guanosine triphosphate analogs ddTTP, ddGTP, and dITP. The nucleotide kinase is found in solution as a 256-kDa complex consisting of ~12 monomers of the gene 1.7 protein. The two molecular weight forms co-purify as a complex, but each form has nearly identical kinase activity. Although gene 1.7 protein does not require a metal ion for its kinase activity, the presence of Mg(2+) in the reaction mixture results in either inhibition or stimulation of the rate of kinase reactions depending on the substrates used. Both the dTMP and dGMP kinase reactions are reversible. Neither dTDP nor dGDP is a phosphate acceptor of nucleoside triphosphate donors. Gene 1.7 protein exhibits two different equilibrium patterns toward deoxyguanosine and thymidine substrates. The K(m) of 4.4 × 10(-4) M obtained with dTTP for dTMP kinase is ~3-fold higher than that obtained with dGTP for dGMP kinase (1.3 × 10(-4) M), indicating that a higher concentration of dTTP is required to saturate the enzyme. Inhibition studies indicate a competitive relationship between dGDP and both dGTP, dGMP, whereas dTDP appears to have a mixed type of inhibition of dTMP kinase. Studies suggest two functions of dTTP, as a phosphate donor and a positive effector of the dTMP kinase reaction.

Helicase-DNA polymerase interaction is critical to initiate leading-strand DNA synthesis.

Interactions between gene 4 helicase and gene 5 DNA polymerase (gp5) are crucial for leading-strand DNA synthesis mediated by the replisome of bacteriophage T7. Interactions between the two proteins that assure high processivity are known but the interactions essential to initiate the leading-strand DNA synthesis remain unidentified. Replacement of solution-exposed basic residues (K587, K589, R590, and R591) located on the front surface of gp5 with neutral asparagines abolishes the ability of gp5 and the helicase to mediate strand-displacement synthesis. This front basic patch in gp5 contributes to physical interactions with the acidic C-terminal tail of the helicase. Nonetheless, the altered polymerase is able to replace gp5 and continue ongoing strand-displacement synthesis. The results suggest that the interaction between the C-terminal tail of the helicase and the basic patch of gp5 is critical for initiation of strand-displacement synthesis. Multiple interactions of T7 DNA polymerase and helicase coordinate replisome movement.

A novel nucleotide kinase encoded by gene 1.7 of bacteriophage T7.

Gene 1.7 of bacteriophage T7 confers sensitivity of both phage T7 and its host Escherichia coli to dideoxythymidine (ddT). We have purified the product of gene 1.7, gp1.7. It exists in two forms of molecular weight 22,181 and 17,782. Only the C-terminal half of the protein is required to confer ddT sensitivity. We show that gp1.7 catalyses the phosphorylation of dGMP and dTMP to dGDP and dTDP, respectively, by using either GTP, dGTP or dTTP as the phosphate donor. Either form of gp1.7 exhibit identical kinase activity as compared with wild-type gp1.7 that contains a mixture of both forms. The K(m) of 70 microM and Kcat of 4.3 s(-1) for dTMP are similar to those found for E. coli thymidylate kinase. However, unlike the host enzyme, gp1.7 efficiently catalyses the conversion of the chain-terminating dideoxythymidylate (ddTMP) to ddTDP. This finding explains the sensitivity of phage T7 but not E. coli to exogenous ddT. Gp1.7 is unusual in that it has no sequence homology to any known nucleotide kinase, it has no identifiable nucleotide-binding motif and its activity is independent of added metal ions. When coupled with nucleoside diphosphate kinase, gp1.7 exponentially converts dTMP to dTTP.

Gene 1.7 of bacteriophage T7 confers sensitivity of phage growth to dideoxythymidine.

Bacteriophage T7 DNA polymerase efficiently incorporates dideoxynucleotides into DNA, resulting in chain termination. Dideoxythymidine (ddT) present in the medium at levels not toxic to Escherichia coli inhibits phage T7. We isolated 95 T7 phage mutants that were resistant to ddT. All contained a mutation in T7 gene 1.7, a nonessential gene of unknown function. When gene 1.7 was expressed from a plasmid, T7 phage resistant to ddT still arose; analysis of 36 of these mutants revealed that all had a single mutation in gene 5, which encodes T7 DNA polymerase. This mutation changes tyrosine-526 to phenylalanine, which is known to increase dramatically the ability of T7 DNA polymerase to discriminate against dideoxynucleotides. DNA synthesis in cells infected with wild-type T7 phage was inhibited by ddT, suggesting that it resulted in chain termination of DNA synthesis in the presence of gene 1.7 protein. Overexpression of gene 1.7 from a plasmid rendered E. coli cells sensitive to ddT, indicating that no other T7 proteins are required to confer sensitivity to ddT.

A randomized controlled trial of the effectiveness of topical amethocaine in reducing pain during arterial puncture.

OBJECTIVE: To determine if topical 4% amethocaine gel can reduce the pain associated with arterial punctures. DESIGN: Randomized, placebo-controlled, double-blinded trial with parallel groups. SETTING: Teaching hospital. PATIENTS: Adults requiring arterial punctures for blood gas estimation as part of routine care. INTERVENTIONS: Four percent amethocaine gel applied for 30 min prior to the radial arterial puncture, compared with a placebo gel. MAIN OUTCOME MEASURES: Pain scored on a visual analog scale from 0 to 100, and heart rate during the procedure. RESULTS: The mean pain score for the amethocaine group was 16.0 (SD, 23.3) and for the placebo group was 20.7 (SD, 18.5). The mean heart rates during arterial puncture were 84.1 beats/min (SD, 10.7) for the amethocaine group, and 83.8 beats/min (SD, 12.7) for the placebo group. These differences were not statistically significant. CONCLUSION: The topical use of 4% amethocaine gel does not reduce the pain associated with arterial puncture.